Rett syndrome (RTT) is a progressive neurodevelopmental disorder. It affects almost exclusively females (Rett, 1966, Wien Med Wochenschr 116:723-6) and it is one of the most common causes of mental retardation in females. RTT is characterized by a dynamically clinical course with four consecutive stages. During Stage I (age 6-18 months), girls cease to acquire new skills; they display decelerating head growth and autistic features such as emotional withdrawal and diminished eye contact. In Stage II (age 1-4 years), affected children lose learned skills such as speech and purposeful hand use. They develop irregular breathing patterns, truncal and gait ataxia/apraxia, and stereotypical hand movements. About half the girls also develop seizures and there is some stabilization of the disease during Stage III (age 4-7 years). Seizures become less frequent during Stage IV (age 5-15 years and older), but motor deterioration continues. Hypoactivity, especially among those who cannot walk, contributes to the frequent development of scoliosis, which can cause the girls to be confined to wheelchairs. Neuropathological features of the Rett patients include a decrease in the cortical thickness as well as a reduction of the cortical neuronal size. A strong reduction of the dendritic arborization has also been described but no further gross morphological features are affected (Armstrong, 2002, Ment Retard Dev Disabil Res Rev 8:72-6). RTT is a X-linked disease with an estimated incidence of 1:10,000 to 1:15,000. Amir et al. (1999) (Nat Genet 23:185-8) have identified mutations in the gene MECP2 as the cause of RTT. The MECP2 gene is subject to X-inactivation. Therefore, heterozygous mutant females are mosaic for MeCP2 deficiency and this is most probably one of the modulating factors influencing the phenotype of the disease. Males meeting the clinical criteria for Rett syndrome have been identified in association with a 47,XXY karyotype and from postzygotic MECP2 mutations resulting in somatic mosaicism. For reference, see L. S. Weaving et al.: Journal of Medical Genetics 2005; 42:1-7; and G. Miltenberger-Miltenyi, F. Laccone: Human Mutation 2003 Volume 22, Issue 2; 107-115.
The MECP2 gene is located on the long arm of the X chromosome at position Xq28 (Adler 1995, Mamm Genome 6(8):491-2). The gene spans 76 kb and is composed of four exons. The MECP2 gene encodes for a protein called methyl-CpG-binding protein 2 (MeCP2), believed to play a pivotal role in silencing other genes. The MeCP2 protein has two isoforms, MeCP2 e1 and MeCP2 e2, formerly called MeCP2B and MeCP2A, respectively (Mnatzakanian et al. 2004, Nat Genet 36:339-41). The isoform e1 is made up of 498 amino acids and isoform e2 is 486 amino acids long. The isoform e1 has a distinctive 21-amino-acid peptide at N terminus including polyalanine and polyglycine tracts. The mRNA of the MECP2 e1 variant has 10-fold greater expression in the brain than that of the MECP2 e2 and it is the most abundant protein isoform in mouse and human brain. MeCP2 is an abundant mammalian protein that selectively binds 5-methyl cytosine residues in symmetrically positioned dinucleotides. CpG dinucleotides are preferentially located in the promoter regions of genes. They represent one of the elements for gene regulation being target for transcriptional silencing factors after DNA methylation.
No successful treatment is yet known to improve the neurological outcome of individuals with Rett syndrome. According to the mutational spectrum of the MECP2 in human (Lam et al. 2000, J Med Genet 37(12):E41; Lee et al. 2001, Brain Dev 23:S138-43) and results from the RTT mouse, there is a general agreement that RTT syndrome is caused by a loss of function of MeCP2. An efficient strategy aiming to restore the MeCP2 activity should be able to compensate the loss of function of MeCP2 in deficient neuronal cells. For reference, see L. S. Weaving et al.: Journal of Medical Genetics 2005; 42:1-7; and G. Miltenberger-Miltenyi, F. Laccone: Human Mutation 2003, Volume 22, Issue 2; 107-115.
Schwarze et al. (1999, Science 285:1569-1572) report that it is possible to deliver biologically active macromolecules into living cells using a TAT domain (transcriptional transactivator protein of human immunodeficiency virus-1). They show the production of a recombinant TAT-β-galactosidase protein and its injection intraperitoneally into mice. They found that the fusion protein was distributed to all tissues including the brain and the fusion protein was biologically active.
WO 00/62067 (Dowdy et al.) report the use of protein transduction (PTD) molecules including the TAT protein domains for targeting therapeutic molecules in the nervous system.
The production of proteins in heterologous expression systems (i.e. human protein in Escherichia coli) requires a corresponding expression vector and the cDNA sequence of the gene of interest. The 64 codons (nucleotide triplets) of the genetic code encode for 20 amino acids and for three translation stop signals. The genetic code is therefore redundant and this means that some amino acids are encoded by more than one codon. Methionine and tryptophane are the only amino acids encoded by one codon, ATG and TGG, respectively, while arginine, leucine and serine are encoded each by six synonymous codons. Because of the degeneration of the genetic code, many alternative nucleic acid sequences encode the same protein. The frequencies of the codons usage vary between the different organisms. These biases can strongly influence the expression of heterologous proteins (Kane, 1995, Curr Opin Biotechnol 6(5):494-500). Codon usage has been identified as the single most important factor in prokaryotic gene expression (Lithwick, 2003, Genome Res 13 (12):2665-2673).
One objective of the present inventors is therefore to provide constructs encoding for biologically active MeCP2 proteins which are capable of entering cells of the nervous system and compensate for the loss of function of MeCP2 of affected neuronal cells.
A therapeutic approach by delivering TAT-recombinant proteins to the brain could have the tremendous advantage of a possible rapid translation from the animal model to patients. Further advantages might be its easily controllable dosage application, very high delivery efficiency, no concerns inherent to the potentially insertional mutagenenesis or clinical side effects due to the immunological reaction against viral proteins as in case of a gene therapy approach. The PTD-protein delivery approach and its further development might allow the treatment of neurogenetic and devastating diseases like Rett syndrome.
Since attempts to express MeCP2 protein constructs using the human cDNA sequence in amounts sufficient for therapeutic purposes were so far unsuccessful, another objective of the present invention is to provide a MeCP2 expression construct that allows an increased production of recombinant MeCP2 protein.